Chapter 4
REGULATION OF ERYTHROPOIETIN
EXPRESSION IN THE NERVOUS SYSTEM: THE HYPOXIA INDUCIBLE FACTOR
Juan C. Chavez and JoAnn M. Gensert
Burke/Cornell Medical Research Institute, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, 785 Mamaroneck Avenue, White Plains, NY 10605,
USA.
Abstract: Hypoxia is associated with a variety of CNS diseases including stroke, traumatic brain injury and spinal cord injury. Induction of erythropoietin (EPO) is a physiological response to hypoxia in oxygen-deprived tissues.
Hypoxia-induced epo gene expression is regulated by the transcriptional activator hypoxia inducible factor (HIF). The epo gene contains a HIF binding site in its 3' untranslated region, and its expression is upregulated concomitantly with HIF activation in a variety of cell culture models as well as in the brains of rodents exposed to hypoxia or pharmacological agents that mimic hypoxia, such as iron chelators and cobalt chloride. Since HIF is a master regulator of oxygen homeostasis in all mammalian cells and controls the expression of a variety of genes required for cellular adaptation to hypoxia (including epo), this review covers the current knowledge about the oxygen sensing mechanism that regulates the activation of HIF under hypoxic conditions. An important challenge for the future is to determine how modulating the activation of HIF with the subsequent expression of EPO can be beneficial for neural survival under stress conditions that involve hypoxia.
Key words: hypoxia, transcription, ischemia, oxygen, neuroprotection.
1. INTRODUCTION
The mammalian central nervous system (CNS) requires a steady supply
of oxygen (O2) to support synthesis of ATP, which is essential to maintain
normal cell homeostasis, in particular neuronal function (Siesjo and Plum,
1971; Siesjo, 1984). When the availability of O2 becomes limited, the CNS
is subject to a detrimental metabolic stress that may cause cell death depending on the duration and severity of the insult (Katsura et al., 1994;
Siesjo, 1981). This hypoxic stress causes the activation of a variety of endogenous compensatory mechanisms aimed at restoring the balance between local oxygen delivery and tissue oxygen consumption. Immediate brain adaptive responses to hypoxia include vasodilation of cerebral arteries and veins resulting in a reversible increase in blood flow (LaManna et al., 1992; LaManna and Harik, 1997); whereas long-term responses involve changes at the level of gene expression. The hypoxia inducible factors (HIFs) are central regulators of these long-term adaptive responses (Semenza, 1998; Semenza, 1999).
Hypoxia inducible factors control the hypoxia-dependent upregulation of a variety of target genes that together facilitate the cellular adaptation to low O2 (Semenza, 2000a). These hypoxia-inducible genes include glucose transporters (Glut-1) and glycolytic enzymes, which will promote metabolic adaptation (Semenza et al., 1994), and angiogenic factors such as the vascular endothelial growth factor (VEGF), which will stimulate formation of new blood vessels and augment tissue O2 delivery (Forsythe et al., 1996;
Semenza, 2003a). In addition, at the systemic level, hypoxia induces the expression of EPO that will enhance red blood cell production and augment O2 carrying capacity (Monge and Leon-Velarde, 1991; Semenza, 1994b).
Hypoxia also induces expression of EPO in the CNS where it functions as a trophic factor promoting cell survival, particularly during severe insults such as stroke and traumatic brain injury (Buemi et al., 2002; Digicaylioglu and Lipton, 2001; Gassmann et al., 2003; Marti, 1996; Marti et al., 1997).
Erythropoietin is the best characterized oxygen-responsive gene and an ideal model to study hypoxia induced gene expression at the molecular level (Bunn et al., 1998). In fact, our current knowledge on the mechanism of oxygen sensing that controls HIF activation and oxygen-regulated gene expression in mammalian cells was acquired primarily through our efforts to understand how EPO expression is increased during hypoxia (Goldberg et al., 1991; Semenza, 1994b; Semenza, 1994a; Wang and Semenza, 1995;
Wang and Semenza, 1996). Hypoxia-induced expression of EPO is controlled primarily at the level of transcription; however, mRNA stabilization contributes significantly to the total amount of EPO message (Ho et al., 1995). Within the epo gene, there is a cis-acting regulatory element that is responsible for its hypoxic induction (Maxwell et al., 1993).
Characterization of this 3' enhancer led to the discovery of HIF, which binds
to a specific region within this enhancer thereby mediating transcriptional
activation of EPO (Maxwell et al., 1993; Semenza and Wang, 1992; Wang
and Semenza, 1995). This discovery was central to the understanding of the
molecular mechanisms regulating hypoxia-induced gene expression. It
4. REGULATION OF EPO EXPRESSION IN THE CNS: HIF 51 unveiled a ubiquitous oxygen sensing mechanism that regulates the
activation of HIF, which not only regulates the expression of EPO but also a broad range of genes that together facilitate the cellular adaptation to low oxygen. This mechanism is highly conserved in vertebrates and can be found also in lower organisms such as Drosophila Melanoghaster and Caenorhabditis Elegans (Bacon et al., 1998; Epstein et al., 2001; Semenza, 2001). This chapter will discuss the role of HIF as a key transcriptional regulator of EPO and the oxygen sensing mechanism that activates HIF in mammalian tissues including the CNS.
2. ERYTHROPOIETIN EXPRESSION IN THE CNS
Systemic EPO is produced mainly in the liver during fetal development
and then in the kidney in adulthood (Zanjani et al., 1981). The expression of
EPO, however, is not restricted to these organs. EPO also is expressed in
testis, uterus, lung, spleen, heart and bone marrow when animals are
subjected to hypoxia (Fandrey and Bunn, 1993; Tan et al., 1991). Moreover,
EPO and EPO receptor (EPOR) are expressed in the developing and mature
central nervous system (CNS) (Digicaylioglu et al., 1995; Fandrey and
Bunn, 1993; Marti et al., 1996). In early fetal stages, EPO expression has
been detected in the periventricular germinal matrix zone, subpial granular
layer, thalamus, hippocampus, lateral geniculate nuclei, cortex and spinal
cord (Dame et al., 2000; Juul et al., 1998). In the adult brain, constitutive
expression of EPO mRNA has been detected in cortical regions, amygdala
and hippocampus - areas particularly susceptible to hypoxic/ischemic insults
(Marti et al., 1996; Marti et al., 1997). At the cellular level, EPO and/or
EPOR have been detected in all major neural cell types. EPO and EPOR
expression in vitro and in vivo in astrocytes, with the receptor robustly
expressed in astrocyte processes surrounding capillaries and in neurons is
well established (Bemaudin et al., 2000; Marti et al., 1996; Masuda et al.,
1994; Siren et al., 2001). Oligodendrocyte expression of EPO and EPOR has
been reported in cells isolated from embryonic rat brain and human CNS
(Nagai et al., 2001; Sugawa et al., 2002). Although expression of EPOR has
been reported for microglial cells isolated from human tissue (Nagai et al.,
2001), as well as for human and rodent endothelial cells in vitro and in vivo
(Anagnostou et al., 1994; Brines et al., 2000; Yamaji et al., 1996), whether
these cells produce EPO remains to be established. As in liver and kidney,
EPO expression in the CNS is also oxygen-sensitive. Albeit, the temporal
pattern of hypoxia-induced EPO expression in CNS differs from that in
kidney and liver; whereas EPO expression in response to hypoxia is transient
in these tissues, EPO expression in the brain is sustained for as long as the
hypoxic stimulus persists (Chikuma et al., 2000). Hypoxia induces EPO in both astrocytes and neurons both at the mRNA and protein levels (Bemaudin et al., 2000; Masuda et al., 1994). Astrocytic and neuronal EPO expression is induced also by desferrioxamine (DFO) and cobalt chloride (C0CI2), two classic pharmacologic agents that mimic hypoxia and cause activation of HIF (Bergeron et al., 2000).
3. THE EPO GENE: PROMOTER REGION AND CIS- ACTING REGULATORY ELEMENTS.
Epo is a single-copy gene located on chromosome 7 in humans and on chromosome 5 in mice (Bunn and Poyton, 1996; Bunn et al., 1998). This gene consists of five exons, four introns and several cis-acting regulatory elements (Fig. 4-1). These DNA regulatory sequences were identified through a transgenic approach in which various DNA fragments of the human epo gene were introduced, with subsequent EPO induction examined in different tissues (Semenza, 1994a). These studies identified specific hypoxic responsive regions of the epo gene for the liver and kidney as well as negative regulatory elements that repressed its expression in a tissue specific manner (Semenza, 1994b; Semenza, 1994a). For further analysis of hypoxia-induced EPO gene expression, two human liver tumor cell lines, HepG2 and Hep3B, were utilized. These cell lines express high levels of EPO when challenged with hypoxia and serve as classical cellular model systems to study the molecular mechanisms of hypoxia-induced EPO expression (Goldberg et al., 1991; Nielsen et al., 1987). Two neuroblastoma cell lines, SH-SY5Y and Kelly, can also be used as models to study hypoxia- induced EPO expression in neural cells. These cell lines produce EPO in response to hypoxia, although they require a more severe hypoxic stress (lower PO2) compared to hepatoma cells (Stolze et al., 2002).
The epo promoter, unlike most promoters, lacks the canonical TATA or
CAAT sequences. Although this promoter is weak, during hypoxia, its
activity increases, synergizing with other cis-acting elements to achieve a
robust induction of epo gene expression in vivo (several hundred- to 1000-
fold) and reporter gene expression in vitro (50- to 100-fold) (Blanchard et
al., 1992; Blanchard et al., 1993; Imagawa et al., 1991). Hypoxia inducible
fissue specific cis-regulatory elements for liver (liver inducible element,
LIE) and for kidney (kidney inducible element, KIE) are located respectively
in the 3'- and 5'- flanking regions of the epo gene. A negatively regulated
liver element (NRLE) is located 3' to the LIE, and a negative regulatory
element (NRE) that represses epo gene expression in non-EPO expressing
cells is located just upstream of the transcription start codon (Semenza et al..
4. REGULATION OF EPO EXPRESSION IN THE CNS: HIF 53 1990; Semenza et al., 1991a). Identification of neural-specific cis-elements has not been elucidated.
3' Enhancer -14 -9.5 -6 -0.4
WHl-OO-ClIhCimE
-118 bp PROiOTER +1
HRE
Figure 4-1. The human epo gene including the promoter and 3' enhancer region (Adapted from Bunn et. al., 1999)
A region known as the 3' enhancer is the most critical hypoxia- responsive cis-element of the epo gene. It is a conserved 40-bp element in the 3'-region, located 120 bp downstream of the polyadenylation site in a region highly conserved between human and mouse sequences (Beck et al., 1991; Pugh et al., 1991; Pugh et al., 1994a; Semenza et al., 1991b; Semenza and Wang, 1992) . This cis-acting DNA element, when linked to a reporter gene and transfected into hepatoma cells, is sufficient to elicit a hypoxia- induced response similar to the EPO response in vivo (Maxwell et al., 1993).
There are three distinct regions of this enhancer: a HIF-1 binding site (HBS), a CACA repeat, and a direct repeat of two steroid receptor half sites (DR-2) (Blanchard et al., 1992; Pugh et al., 1994b; Semenza and Wang, 1992). The HBS consists of a highly conserved 5' portion element (13 bp) with a consensus sequence CA/(G)CGTGCT. Electromobility shift assays using double stranded oligonucleotides were used to demonstrate that a nuclear protein (HIF-1) from hepatoma cells binds this sequence only when cells are subjected to hypoxia; this binding is essential for hypoxic EPO induction (Semenza and Wang, 1992). In addition, a constitutive protein complex formed by ATF-1 and CREB-1 was identified (Kvietikova et al., 1995).
However, the role of this complex in hypoxia-regulated EPO expression has not yet been elucidated. A second element of the 3' enhancer in the human gene consists of three CA repeats (Pugh et al., 1991; Pugh et al., 1994b).
Although not highly conserved, it is necessary for hypoxic induction of EPO
as well as of other HIF-1 target genes. Which protein(s) recognize and bind
this CA repeat site is not yet known. The third element of the epo enhancer, DR-2, is located 3' to the other two and is absolutely required for the induction of EPO expression during hypoxia (Blanchard et al., 1992). Direct repeat steroid receptor half sites are known to bind a variety of hormone nuclear receptors. However, none of the classic ligands for the direct repeats half sites was shown to modulate epo expression through the DR-2 binding sites (Blanchard et al., 1992). Screening for orphan receptors has identified a protein called hepatocyte nuclear factor-4a (HNF4a). In the Hep3B hepatoma cell line, HNF4a is required for the induction of EPO during hypoxia (Galson et al., 1994). The neuroblastoma cell lines, SH-SY5Y and Kelly, which show robust upregulation of EPO in response to hypoxia, however, lack HNF4a and other HNF4 isoforms (Stolze et al., 2002).
Therefore the DR-2 sites and HNF4a may play a role in tissue specific expression of EPO. Since the DR-2 sites are required for hypoxic induction of EPO, a yet unknown factor must substitute HNF-4 and regulate EPO expression in neural cells.
As noted previously, astrocytes and neurons upregulate EPO in vitro and in vivo not only in response to hypoxia, but also in response to DFO and C0CI2. As these two agents are well-characterized HIF-1 activators, it is likely that HIF-1 is a central mediator of hypoxia-induced EPO expression in neural cells. Although details about other regulatory elements that contribute to hypoxia-induced EPO expression in the CNS are not known, the oxygen sensing mechanism that regulates HIF-1 activation is well characterized and it is highly conserved in all tissues including the CNS.
4. HYPOXIA INDUCIBLE FACTOR (HIF)
HIF is a heterodimer formed by two subunits that belong to the PAS (Per, Amt, Sim) family of basic helix-loop-helix (bHLH) transcription factors;
these subunits are designated HIF-a and HIF-p. The expression of the HIF-a
subunits is regulated by oxygen levels, whereas the HIF-|3 subunits, also
known as arylhydrocarbon receptor nuclear translocator (ARNT), are
constitutive nuclear proteins that dimerize with other bHLH-PAS
transcription factors (Semenza et al., 1997; Semenza, 1998; Wang and
Semenza, 1995; Wang et al., 1995). Currently, three HIF-a subunits (HIF-
l a , HIF-2a/EPASl and HIF-3a) as well as three HIF-P subunits (HIF-
1|3/ARNT1, ARNT2 and ARNT3) are known (Semenza, 1999; Talks et al.,
2000). The most widely expressed alpha subunit in mammalian tissues is
HIF-la; indeed most of our knowledge about HIF comes from studies of the
mechanism regulating the expression of HIF-la protein and the
transcriptional activity of the HIF-1 complex (HIF-la/HIF-ip heterodimer).
4. REGULATION OF EPO EXPRESSION IN THE CNS: HIF 55 The other HIF-a subunits are regulated by a similar mechanism although
they appear to have more specialized and tissue specific functions (Semenza, 1999). During normoxia, the HIF-la protein is constitutively expressed, but it is rapidly destroyed by the ubiquitin-proteosome system, such that almost no HIF-la protein accumulates (Huang et al., 1998; Salceda and Caro, 1997;
Salceda and Caro, 1997). Under hypoxic conditions, degradation of the HIF- l a subunit is prevented, allowing HIF-la to accumulate within the nucleus where it dimerizes with HIF-1(3 forming the HIF-1 transcriptional complex (Jewell et al., 2001; Salceda and Caro, 1997). HIF-1 binds to a consensus DNA sequence A/(G)CGTG within the hypoxia response elements (HRE) of numerous hypoxic responsive target genes that include EPO, glycolytic enzymes, angiogenic factors, and glucose transporters, among others (Semenza, 1999; Wang and Semenza, 1993; Wang et al., 1995).
4.1 Molecular mechanism of HIF-1 activation during hypoxia: oxygen-sensing mechanism.
Regulation of HIF by O2 is mediated by two distinct pathways that involve enzymatic trans-4-hydroxylation of two proline residues and the P- hydroxylation of an asparagine residue in the HIF-a subunits. Prolyl hydroxylation the HIF-a subunit is carried out by an enzyme encoded by the egg-laying abnormal-9 (Egl-9) gene in C. Elegans and D. Melanogaster.
The mammalian homologues are named egg laying nine 1 (EGLNl), EGLN2 and EGLN3, also called prolyl hydroxylase domain-containing proteins PHD2, PHDl, and PHD3, respectively (Epstein et al., 2001).
Degradation of HIF-a under normoxic conditions is triggered by post- translational hydroxylation of the conserved proline residues, Pro-402 and Pro-564, within a region of the HIF-a protein known as the oxygen- dependent degradation (ODD) domain (see below and Fig. 4-3). The hydroxylated proline residues in this domain are recognized by the product of the von Hippel-Lindau tumor suppressor gene (p VHL), which acts as the recognition component of a multiprotein ubiquitin E3 ligase complex, thus targeting HIF-a to ubiquitin-mediated proteolysis in the proteosome (Ivan et al., 2001; Jaakkola et al., 2001; Masson et al., 2001; Maxwell et al., 1999).
This regulatory post-translational modification is inherently oxygen
dependent, since the hydroxyl group is derived from molecular oxygen
(Bruick and McKnight, 2001; Epstein et al., 2001; Ivan et al., 2001). The
prolyl hydroxylation also requires the cofactors 2-oxoglutarate, vitamin C
and iron (II). The requirement of iron explains the hypoxic-mimetic effects
of iron chelators (such as deferoxamine mesylate) and iron antagonists (such
as cobalt chloride). Under low O2 conditions, or in the presence of iron
chelators, HIF-a is not hydroxylated by PHDs and therefore HIF-a is neither
recognized by pVHL nor targeted for degradation by the proteosome (Fig. 4- 2). As a result, HIF-a accumulates in the nucleus and is available to dimerize with HIF-p subunits to form the active HIF complex that activates transcription of target genes including EPO.
HYPOXIA
. ' H I F - i . : ;
fHiFii;:;; -^io
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h-lct J * • " * * * ' • ' '
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" . EPO H I F - I c i j ,. and other H I F - 1 P 1 "• '••••"HiF-1 target genes Active HIF-1
complex
Reoxygenation (min)
N H 5 10 15 30 60 .,PP _
N= 2 1 % Oj H = 1 % 0 2
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HIF-la
Figure 4-2. Regulation of HIF-la by O2 level. In cultured cortical neurons, HIF-la protein accumulation is regulated also by O2 tension as shown by western blot analysis.
Hypoxia affects not only HIF-a protein stability but also the transcriptional activity of the HIF complex (Bruick and McKnight, 2002;
Jiang et al., 1997; Pugh et al., 1997). The three alpha subunits contain two transactivation domains (TAD) that interact with transcriptional co- activators essential for gene expression (Figure 3). The amino terminal TAD (N-TAD, aa 531-575) overlaps with the ODD domain (aa 401-603). The carboxy terminal TAD (C-TAD, aa 786-826) is independent of the ODD domain and is able to recruit co-activators such as p300/CBP under hypoxic conditions only (Ema et al., 1999; Gu et al., 2001; Semenza, 2002). The regulation of this C-TAD involves an oxygen-dependent hydroxylation of a conserved asparagine residue (Asn-803 in HIF-1 a and Asn-851 in HIF-2a).
This hydroxylation is catalyzed by a novel asparaginyl hydroxylase that was
described previously as a factor inhibiting HIF-1 (FIH-1). This asparaginyl
hydroxylase is also a member of the 2-oxoglutarate- and iron-dependent
dioxygenase superfamily; hence it is inhibited by hypoxia (Lando et al.,
2002;Mahon et al., 2001). When the asparagine residue is hydroxylated, the
C-TAD cannot interact with the transcriptional coactivator p300/CBP and
the HIF-1 transcriptional activity is reduced.
4, REGULATION OF EPO EXPRESSION IN THE CNS: HIF 57 Histone acetylation state is also a factor that affects HIF-1 transcriptional activity. In addition to promoting ubiquitination and degradation of HIF-la, VHL forms a ternary complex with HIF-la and the co-repressor FIH-1.
Both VHL and FIH-1 recruit histone deacetylases that may contribute to the loss of HIF-1 transcriptional activity under non-hypoxic conditions (Mahon etal.,2001).
bHLHi T | A r p A s 1 B I r
Pro-402 Pro-564 ASii-803
- L , I , mm I i
Dimerization and DNA binding
401 O D D 603
Regulatory domain Transactivation
C-TAD
HYPOXIA
bHLH 1 1 1 A 1 PAS ! B j 1
Dimerization and DNA binding
Pro-402 Pro-564
1 li N-TAD j
: ; :•:
401 O D D 603
Regulatory domain Transactivation
ASn-803 H'^-?'-s-- C-TAD ;•;
Figure 4-3. Oxygen mediated post-translational modifications of HIF-la subunit.
In addition to hydroxylation, a new post-translational modification of the
HIF-la subunit has been described, where a novel HIF-la protein acetyl
transferase called ARDl was shown to acetylate the lysine 532 residue of
HIF-la. This acetylation enhances the interaction of HIF-la with VHL,
thereby augmenting the subsequent HIF-la ubiquitination and proteosomal
degradation. Moreover, this report showed that the expression of ARDl is
reduced by hypoxia, consistent with the accumulation of HIF-la during low
oxygen conditions (Jeong et al., 2002). Collectively, these findings
demonstrate that during hypoxia, HIF-1 is regulated by a carefully controlled
signal transduction pathway in which a group of hydroxylases act as putative
oxygen sensors by way of their requirement of oxygen for activity. These
hydroxylases catalyze unique oxygen dependent posttranslational
modifications of the HIF-la subunit that control its degradation and regulate
HIF transcriptional activity.
4.2 Regulation of prolyl hydroxylases expression
Little is known about the expression and functions of HIF-1 prolyl hydroxylases in the CNS. However, the regulation of HIF by the PHDs known to date, PHDl, PHD2, and PHDS, has been studied in vitro (in chemical assays) and in the context of a variety of non-neural cell types. All three of the PHD isoforms hydroxylate HIF-a peptides in vitro (Bruick and McKnight, 2001; Epstein et al., 2001) and contribute to the regulation of HIF in a variety of cell contexts (Appelhoff et al., 2004; Hirsila et al., 2003).
Furthermore, when overexpressed in cells, all three PHDs can suppress HRE-mediated reporter gene activity (Huang et al., 2002; Metzen et al., 2003). Despite distinct intracellular localization patterns of exogenous PHDs - PHDl is exclusively nuclear, PHD2, mainly cytoplasmic, and PHD3, both cytoplasmic and nuclear - each of the PHDs can regulate nuclear HIF-la during hypoxia (Metzen et al., 2003). Metzen and colleagues also showed that endogenous PHD2 mRNA and PHD3 mRNA are hypoxia-induced.
However, during normoxia, a dominant role for endogenous PHD2 has been demonstrated, in a variety of non-neural cell lines (Berra et al., 2003).
Furthermore, PHD2 has a greater influence on HIF-la than on HIF-2a;
whereas for PHD3, the opposite pattern emerges (Appelhoff et al., 2004).
Admittedly, variations in expression levels of endogenous PHD isoforms appear to be cell type- and culture condition- dependent, warranting investigation of PHDs in neural cell types. To further understand HIF regulation of EPO in the brain, study of these critical HIF regulators, the PHDs, in a neural cell context is essential. These observations suggest that a novel feedback mechanism for adjusting hypoxia-induced gene expression exist that involves regulation of PHD expression.
In a recent study, Nakayama et al. had identified a novel mechanism that regulates the availability of PHDl and PHD3 and consequently affects the abundance of HIF-la (Nakayama et al., 2004). This study showed that PHDl and PHD3 protein levels are regulated by members of the E3 ubiquitin ligase family Siah2 and Siahla. These E3 ligases mediate the ubiquitination and the proteosome-dependent degradation of PHD 1/3 during hypoxia. Generally, PHD activity is diminished during hypoxia, although even at low oxygen concentrations, residual PHD activity may persist. To overcome this residual activity, an additional mechanism of PHD regulation at the protein level is required during hypoxia to facilitate HIF-1 activation and upregulation of hypoxia-responsive genes including EPO (Simon, 2004).
Regulation of PHD availability is therefore another step in the complex
pathway that regulates HIF activation.
4. REGULATION OF EPO EXPRESSION IN THE CNS: HIF 59
4.3 Alternate mechanisms stabilize HIF-la under normoxic conditions
As noted above, DFO and C0CI2 are classic pharmacological agents that mimic some cellular hypoxic responses, including activation of HIF-1.
Numerous studies have shown that a variety of iron chelators including mimosine and DFO are potent activators of HIF since PHD activity requires iron (Ivan et al., 2002; Wamecke et al., 2003). In addition, the divalent transition metal Cobalt (Co^^) has been shown to induce HIF-la protein accumulation under normoxic conditions. Although it was initially proposed that Co^^ acts by displacing iron, a recent report demonstrated that Co^^
induces HIF-la protein accumulation by disrupting the interaction between hydroxy lated HIF-la and VHL, consequently preventing HIF-la ubiquitination and proteosomal degradation (Yuan et al., 2003). Both DFO and C0CI2 have been used successfully to activate HIF and the expression of HIF target genes in neonatal rat brain and adult mouse and rat brain (Bergeron et al., 2000) and were effective in reducing brain injury associated with ischemia in different animal models (Sharp and Bemaudin, 2004).
HIF-la protein expression, HIF-1 DNA binding activity and HIF-1 target gene expression under non-hypoxic conditions are induced also by a variety of growth factors and cytokines, including epidermal growth factor (EOF), fibroblast growth factor 2 (FGF-2), insulin, insulin-like growth factor 1 and 2 (IGF-1, IGF-2), tumor necrosis factor-a (TNFa), interleukin-lp and angiotensin II (Semenza et al., 2000; Semenza, 2000c; Semenza, 2000b).
These growth factors and cytokines bind their cognate receptor tyrosine kinases and activate a variety of signaling pathways, including the phosphatidylinositol 3-kinase (PI3K), the serine-threonine protein kinase Akt (protein kinase B), the mammalian target of rapamycin (mTOR, also known as FRAP) and the ERK/MAPK pathway (Semenza, 2000c; Semenza, 2003b). Many of these pathways have been implicated in the growth factor mediated activation of HIF-1 in a variety of cell lines.
In the CNS, IGF-1 is so far the only growth factor that has been shown to activate the HIF pathway. Interestingly, IGF-1, IGF-2 and insulin can stimulate EPO production in primary cultured astrocytes (Masuda et al., 1997). In a model of global cerebral ischemia in rats, IGF-1 mediates in part the activation of HIF-1 independently of hypoxia. Moreover, exogenous systemic or intra-cerebroventricular infusion of IGF causes HIF-la accumulation and expression of HIF-1 target genes including EPO (Chavez and LaManna, 2002). In a recent study, Lopez-Lopez et al. showed that IGF-
1 induces the growth of cultured brain endothelial cells through activation of
HIF and its target gene, VEGF. This study also showed that systemic
injection of IGF-I in adult mice increases brain vessel density (Lopez-Lopez
et al., 2004). Taken together these data support the role of IGF-1 as an important regulator of HIF activation in the adult CNS.
4.4 Physiologic role of HIF-1 in the CNS
Hypoxia inducible factor-1 has a critical physiological role in the CNS; it is absolutely required for normal development. Mouse embryos that lack HIF-la die at midgestation, with multiple cardiovascular defects and mesenchymal cell death (Yu et al., 1999) . Also, HIF-la is necessary for normal development of the brain. In a mouse model of neural cell specific HIF-la-deficiency, animals were viable and reached adulthood; however, they developed hydrocephalus and showed a marked reduction in brain mass (Tomita et al., 2003). In the adult brain, HIF-1 a is expressed constitutively (Stroka et al., 2001) and is further induced by hypoxia in neurons, astrocytes, ependymal cells and possibly endothelial cells (Chavez et al., 2000b).
Whether HIF-1 is activated in microglia and oligodendrocytes is not known.
In contrast, HIF-2a seems to be induced preferentially in glia and endothelial cells, but not in neurons (Wiesener et al., 2003). A recent study suggests that expression of HIF-la and HIF-2a results in the induction of different HIF target genes. In particular, the expression of EPO seems to depend primarily on HIF-2a activation (Ralph et al., 2004). In the brains of rodents exposed to hypoxia, HIF-la accumulation correlates with the upregulation of HIF regulated genes that include glucose transporters (Glut-
1), glycolytic enzymes, pro-angiogenic factors (VEGF and Flt-1) and EPO (Bergeron et al., 1999;Chavez et al., 2000a) (Fig. 4-4). Similarly, in a variety of cerebral ischemia models, EPO is upregulated at the mRNA level (Bergeron et al., 1999;Bergeron et al., 2000;Sharp et al., 2001;Sharp and Bemaudin, 2004). Taken together, the in vivo and in vitro data implicate HIF as a critical mediator of EPO expression in the CNS.
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